The applicants have synthesized and characterized new fluorescent dyes, called FM dyes, which stain living nerve terminals in an activity- dependent fashion. The dyes appear to work by labeling the membranes of recycled synaptic vesicles. In frog motor nerve terminals, for example, staining with FM dyes produces a series of bright, bead-like fluorescent spots along the terminals, each spot marking a cluster of synaptic vesicles. The spots, which are 1-2 um in diameter, persist indefinitely, unless the nerve is stimulated, in which case they dim and disappear, evidently reflecting release of dye by exocytosis from labeled vesicles. The FM dyes offer new opportunities for studying synaptic function. In preliminary work, the applicants have used them to monitor optically synaptic activity in more than a dozen different biological preparations.The applicants have also used them to study the cell biology of synaptic vesicle trafficking, which is the subject of this proposal. The applicant will use the dyes to study synaptic vesicle recycling in living nerve terminals, addressing mechanisms by which vesicles are held in resting terminals, and mechanisms by which they are mobilized and transported to the presynaptic membrane. The applicants will also study processes that occur after vesicle exocytosis, for example, how and when membrane is reinternalized, and how vesicles are regenerated in time and space. In other projects, the applicants will study the mechanism of action of botulinum toxin, testing the hypothesis that it immobilizes synaptic vesicles. The applicants will explore the use of new preparations to combine optical and electrophysiological (patch clamp/capacitance measurements) techniques to study exocytosis and vesicle recycling. The rationale for this work is simple: Synaptic function underlies all integrative processes in the nervous system, and nearly all therapeutic drugs work at synapses. Knowledge of mechanisms, which guide synaptic vesicles as they repeatedly cycle through rounds of exocytosis and endocytosis, will be useful for understanding broader aspects of nervous system function in health and disease.